Flaviviruses are a genus of related positive-stranded enveloped RNA viruses that significantly impact human health, including dengue (DENV), Zika (ZIKV). West Nile (WNV), Japanese encephalitis (JEV), and yellow fever (YFV) viruses. The discovery of host factors critical for viral infection reveals new aspects of cell biology, intricate virus-host relationships, and potential targets for antiviral therapeutics. After entering cells and fusing in the acidified endosome, the flavivirus RNA genome penetrates into the cytoplasm and is then translated into a polyprotein and processed at the endoplasmic reticulum (ER). In addition to utilizing many functions of the ER for protein production, flaviviruses extensively remodel ER membranes to create a niche for RNA- dependent RNA replication. The ER also is the site for flavivirus assembly, which enables the production and secretion of new infectious viruses. Thus, the ER serves as a central point for orchestrating many of the essential steps in the flavivirus infection life cycle. Despite this, little is known about the host factors and molecular mechanisms at the ER that are required for optimal translation and processing of the viral proteins or for the assembly of the replication niche. We recently have performed several genetic screens to identify important components in this process. Our genome-wide RNAi screen with WNV in insect cells validated 18 genes associated with ER biology that promote infection. Our CRISPR/Cas9 gene-editing screen in human cells with WNV also identified 12 ER-associated genes. These screens converged on ER-resident proteins as being critical for WNV infection and included genes associated with ER-translocation and signal peptide processing, ER-associated degradation (ERAD), protein glycosylation, protein folding and lipid metabolism. Indeed, infection of WNV, ZIKV, JEV, DENV, and YFV all required specific subunit components of the host signal peptidase complex (SPCS) for processing of the viral polyprotein, the production of viral glycoproteins and thus generation of nascent virions. The objective of this proposal is to define the molecular mechanisms by which flaviviruses use specific ER-associated host proteins to promote viral translation, polyprotein processing, RNA replication, and/or assembly. Aim 1 will define the mechanism by which the ER translocon promotes polyprotein translation and processing while Aim 2 will dissect the role of ER-associated decay (ERAD) in promoting flavivirus replication. Our long-term goal is to determine the mechanisms by which flaviviruses exploit the ER for their replication, as this will reveal both fundamental aspects of virology as well as new avenues for antiviral therapeutics.